CN119040442B - A PCR amplification method and kit thereof - Google Patents
A PCR amplification method and kit thereof Download PDFInfo
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- CN119040442B CN119040442B CN202411543601.7A CN202411543601A CN119040442B CN 119040442 B CN119040442 B CN 119040442B CN 202411543601 A CN202411543601 A CN 202411543601A CN 119040442 B CN119040442 B CN 119040442B
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- 238000012408 PCR amplification Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 23
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 63
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 63
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 33
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 230000037048 polymerization activity Effects 0.000 claims abstract description 15
- 150000003839 salts Chemical class 0.000 claims abstract description 6
- 108010006785 Taq Polymerase Proteins 0.000 claims description 34
- 239000002738 chelating agent Substances 0.000 claims description 34
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 23
- 229910001424 calcium ion Inorganic materials 0.000 claims description 23
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical group OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 10
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- 230000003321 amplification Effects 0.000 abstract description 35
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- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 5
- 239000013522 chelant Substances 0.000 description 5
- 239000000174 gluconic acid Substances 0.000 description 5
- 235000012208 gluconic acid Nutrition 0.000 description 5
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 5
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- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 229920000388 Polyphosphate Polymers 0.000 description 3
- 101000717237 Tobacco streak virus (strain WC) RNA-directed RNA polymerase 2a Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 235000015165 citric acid Nutrition 0.000 description 3
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- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000001205 polyphosphate Substances 0.000 description 3
- 235000011176 polyphosphates Nutrition 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
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- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000589596 Thermus Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
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- 230000009920 chelation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 238000000338 in vitro Methods 0.000 description 2
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- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000605059 Bacteroidetes Species 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 241000316914 Prococcus Species 0.000 description 1
- 241001237851 Thermococcus gorgonarius Species 0.000 description 1
- 241001235254 Thermococcus kodakarensis Species 0.000 description 1
- 241000589500 Thermus aquaticus Species 0.000 description 1
- 241000589499 Thermus thermophilus Species 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229910001448 ferrous ion Inorganic materials 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
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Abstract
本发明提供一种PCR扩增方法及其试剂盒，该方法在配制PCR扩增反应体系时，先将含有金属离子的盐与用于PCR扩增的DNA聚合酶溶液进行预混，使得所述金属离子与所述DNA聚合酶结合，占据镁离子与所述DNA聚合酶的结合位点，封闭所述DNA聚合酶聚合活性，在PCR扩增时加入镁离子竞争结合所述DNA聚合酶。本发明提供了一种新的提高DNA聚合酶特异性扩增的方法，与现有技术相比，该方法简单，成本低。
The present invention provides a PCR amplification method and a kit thereof. In the method, when preparing a PCR amplification reaction system, a salt containing metal ions is premixed with a DNA polymerase solution used for PCR amplification, so that the metal ions bind to the DNA polymerase, occupy the binding site of magnesium ions and the DNA polymerase, block the polymerization activity of the DNA polymerase, and add magnesium ions to compete for binding to the DNA polymerase during PCR amplification. The present invention provides a new method for improving the specific amplification of DNA polymerase. Compared with the prior art, the method is simple and low in cost.
Description
Technical Field
The invention relates to the field of nucleic acid amplification, in particular to a PCR amplification method and a kit thereof.
Background
The polymerase chain reaction (Polymerase Chain Reaction, PCR) technology is born in the middle of the 80 s of the last century, and is rapidly developed into a rapid and high-sensitivity method for in vitro analysis of nucleic acid after being proposed by the United states Kary Mullis in 1985, and is one of the most important technical inventions in the field of molecular biology. The principle is that DNA polymerase completes the amplification process of the appointed DNA fragment in vitro in a proper buffer solution under the participation of four deoxyribonucleotides (dNTPs), magnesium ions (Mg 2+), specific primers and templates, namely, specific amplification and enrichment of the specific nucleic acid fragment are realized through enzymatic reaction.
The reaction procedure of general PCR comprises steps of pre-denaturation, annealing, extension and the like (annealing and extension can be combined sometimes), and the reaction temperature is above 55 ℃, so that the heat-resistant DNA polymerase is a core component of a PCR reaction system, and the performance of the heat-resistant DNA polymerase determines the sensitivity, the specificity and the amplification success rate of PCR detection.
The thermostable DNA polymerases currently found belong to either group A or group B. Group a is from eubacteria such as Taq (Thermus aquaticus), tth (Thermus themmophilus), tfl (Thermus flavus), and Tfi (Thermus filfomis), and group B thermostable DNA polymerases are from archaebacteria, commonly including Pfu (Prococcus furiosus) and KOD (Thermococcus kodakaraensis) and Tgo (Thermococcus gorgonarius). The heat-resistant DNA polymerase has the highest activity at a higher temperature, but the polymerization activity of the DNA polymerase still exists at a lower temperature, so that in the initial stage of the PCR reaction, as the quantity of the primer is far higher than that of the template, the primer mismatch or the phenomenon that the primer and the template are subjected to some nonspecific pairing easily occurs in the heating process of an instrument, the primer dimer and nonspecific products are formed by extension under the action of the DNA polymerase, and the nonspecific products can be used as the template to continue to amplify in a PCR cycle, so that the nonspecific products are continuously amplified and accumulated, and the amplification of a target fragment is seriously interfered, so that the amplification efficiency of the target product is low and even cannot be amplified.
In order to solve the problems, the prior art overcomes the defects of the prior DNA polymerase by wax isolation, gene mutation, chemical modification, antibody modification, aptamer or covalent bond binding modification of the DNA polymerase, namely, inhibiting or reducing the activity of the DNA polymerase under the low-temperature condition. Wherein, the amplification by using the hot start DNA polymerase is a main method for improving the specificity of PCR amplification and the sensitivity of amplification. At low temperatures (typically below 55 ℃), antibodies bind specifically to DNA polymerase, blocking its activity, and at high temperatures, antibodies irreversibly dissociate from the enzyme, releasing the activity. The activity of DNA polymerase at normal temperature is blocked by the DNA polymerase antibody, so that non-specific amplification caused by primer mismatch is avoided, and the amplification specificity is improved.
However, the combination of the antibody and the enzyme is easy to be influenced by environment, the invalid blocking is easy to occur, the types of the DNA polymerase mutant are more, the adaptive antibody which can be blocked with high efficiency is difficult to find, and meanwhile, a series of processes such as animal immunization, feeding, protein extraction and purification are required for antibody production, so that the period is longer, the cost is higher, and the cost of PCR reaction is increased.
In PCR amplification, magnesium ion (Mg 2+) acts as a cofactor for DNA polymerase activity, facilitating DNA polymerase binding to dNTPs. The magnesium ion (Mg 2+) at the enzyme active site can catalyze the formation of phosphodiester bonds of the 3' -OH of the primer with the phosphate group of dntps. In addition, mg 2+ is capable of stabilizing the negative charge on the phosphate backbone, thereby promoting the formation of a complex of the primer and the DNA template, facilitating DNA polymerase binding, and thus promoting DNA polymerase polymerization activity. In the absence of magnesium ion (Mg 2+) or in the absence of binding of DNA polymerase to magnesium ion (Mg 2+), no polymerization occurs.
Chelating agents, also known as metal blocking agents, water softeners, and the like, are ligands containing two or more electron donating atoms in the structure. Chelating agents are broadly classified into complex metal chelating agents (ethylenediamine tetraacetic acid (EDTA), ethylene Glycol Tetraacetic Acid (EGTA), aminotriacetic acid (also known as nitrilotriacetic acid NTA), diethylenetriamine pentaacetic acid (DTPA), citric Acid (CA), tartaric Acid (TA), gluconic Acid (GA), etc.), organic polyphosphonic acids and inorganic polyphosphates. The chelating agent has a sequence of chelating of metal ions, and any reaction of metal ions with chelating agents to form chelates is a displacement reaction, i.e. surrounding water molecules coordinated to the metal ions are displaced by the chelating agent. The stability constant of the chelate is the main basis for determining the sequence of the chelation reaction. In solutions containing only one chelating agent but a plurality of metal ions, the chelation reaction generally occurs preferentially between the chelating agent and the metal ion having a large chelate stability constant. If a chelating agent with a larger stability constant is added to other solutions already containing a certain metal chelate, the metal ions will be displaced. In contrast, if the stability constant of the newly added chelating agent is smaller than that of the original chelating agent, the substitution reaction does not occur.
Disclosure of Invention
In order to solve the technical problems, the PCR amplification method comprises the steps of firstly premixing a salt containing metal ions with a DNA polymerase solution for PCR amplification when preparing a PCR amplification reaction system, so that the metal ions are combined with the DNA polymerase, occupy the combining sites of magnesium ions and the DNA polymerase, seal the polymerization activity of the DNA polymerase, and adding magnesium ions to compete for combining with the DNA polymerase during PCR amplification so as to restore the activity of the DNA polymerase for PCR amplification, wherein the combining sequence of the metal ions and the DNA polymerase is lower than that of the magnesium ions and the DNA polymerase.
In one embodiment, the metal ion is at least one of copper ion, zinc ion, nickel ion, calcium ion, manganese ion, cobalt ion, iron ion, or ferrous ion, and/or the DNA polymerase is at least one of Taq DNA polymerase, tth DNA polymerase, tfl DNA polymerase, tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, or Tgo DNA polymerase.
In one embodiment, the DNA polymerase is mixed with the metal ion in a reaction ratio of 10U/. Mu.L of DNA polymerase to 50 mM to 300mM gold ions.
In one embodiment, the DNA polymerase is Taq DNA polymerase, the metal ion is calcium ion, and the ratio of DNA TAQ DNA polymerase to calcium ion is 10U/. Mu.L 80-100 mM.
In one embodiment, the molar ratio of the calcium ions to the magnesium ions is no greater than 1:1.
In one embodiment, the molar ratio of the calcium ions to the magnesium ions is 1:1 to 1:2.
In one embodiment, a metal chelator is also added during PCR amplification, the chelator chelating the metal ion in a better order than the chelator chelating the magnesium ion.
In one embodiment, the chelating agent is at least one of ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, aminotriacetic acid, diethylenetriamine pentaacetic acid, citric acid, tartaric acid, gluconic acid, organic polyphosphonic acid, inorganic polyphosphates.
In one embodiment, the metal ion is a calcium ion and the chelating agent is EGTA, the molar ratio of the calcium ion to the chelating agent being no greater than 1:2.5.
In one embodiment, the molar ratio of the calcium ions to the chelating agent is 1:1 to 1:1.5.
In one embodiment, the present invention provides a PCR amplification kit for use in the above method.
In the present invention, the ratio of the DNA polymerase to the metal ion may be any of 10U/. Mu.L, 50 mM, 10U/. Mu.L, 80 mM, 10U/. Mu.L, 100 mM, 10U/. Mu.L, 150 mM, 10U/. Mu.L, 250 mM, 10U/. Mu.L, 300 mM, and the source of magnesium ion (Mg 2+) may be selected from one of magnesium chloride, magnesium sulfate and magnesium acetate, and the amount thereof may be 5mM,10 mM,20 mM,30 mM,40 mM.
The metal salt and the DNA polymerase are premixed according to a certain proportion, and occupy the binding site of magnesium ion (Mg 2+) and the DNA polymerase, so that the polymerization activity is blocked, and the amplification of the DNA polymerase under the low-temperature condition is inhibited. And adding a metal ion chelating agent into the PCR reaction buffer solution, and chelating other metal ions combined on the DNA polymerase by the metal ion chelating agent in PCR amplification circulation so as to deblock, thereby realizing the specific amplification of the template.
The invention provides a chelating agent for chelating metal ions, which is added during PCR amplification and at least contains one of complex metal chelating agents (ethylenediamine tetraacetic acid (EDTA), ethylene Glycol Tetraacetic Acid (EGTA), aminotriacetic acid (also called nitrilotriacetic acid NTA), diethylenetriamine pentaacetic acid (DTPA), citric Acid (CA), tartaric Acid (TA), gluconic Acid (GA) and the like), organic polyphosphonic acid and inorganic polyphosphates. The ratio of the metal salt to the chelating agent is one of 1:1,1:1.5,1:2,1:2.5,1:3 and 1:4.
In the invention, a method for improving the specificity and the sensitivity of the PCR reaction, which has low cost and convenient use, is found by utilizing the mechanism that the magnesium ions (Mg 2+) are combined with the DNA polymerase in the PCR reaction to exert the polymerization activity of the DNA polymerase and the sequential chelating property of chelating agents on different metal ions.
Compared with the prior art, the method is simple and low in cost, the metal salt and the DNA polymerase are premixed according to a certain proportion to block the polymerization activity of the DNA polymerase, and a certain amount of magnesium ions (Mg 2+) are added to compete for binding with the DNA polymerase to block during PCR amplification, or chelating agents are used for chelating the metal ions for blocking the polymerization activity to block, so that the specific amplification occurs, and the probability of non-specific amplification is reduced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of amplification after premixing CaCl 2 and Taq DNA polymerase, and in PCR amplification, mg 2+ is in competition with Taq DNA polymerase, wherein a control group Taq sample is Taq DNA polymerase directly added into a system for PCR amplification, and experimental groups Taq-Ca1, taq-Ca2, taq-Ca3, taq-Ca4 and Taq-Ca5 sample are subjected to PCR amplification by using Taq DNA polymerase and CaCl 2 for blocking after incubation and activity;
FIG. 2 is a graph showing the results of amplification after adding different concentrations of Mg 2+ to compete for binding to Taq DNA polymerase during PCR amplification after premixing CaCl 2 and Taq DNA polymerase, wherein a control group Taq sample is prepared by directly adding Taq DNA polymerase into a system for PCR amplification, and an experimental group Taq-Ca1+ MgCl 2 sample with different concentrations is prepared by performing PCR amplification after using Taq DNA polymerase and CaCl 2 to incubate blocking activity and using different concentrations of Mg 2+ for deblocking;
FIG. 3 is a schematic diagram showing amplification after adding EGTA chelate Ca 2+ for deblocking after premixing CaCl 2 and Taq DNA polymerase used in the invention and PCR amplification, wherein the control group Taq sample is a Taq DNA polymerase directly added system for PCR amplification, and the experimental group Taq-Ca1+ EGTA sample with different concentrations is subjected to PCR amplification after deblocking by EGTA chelate Ca 2+ after incubation of blocking activity with CaCl 2 by using Taq DNA polymerase.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. In the following examples, unless otherwise indicated, all methods conventional in the art are described.
Example 1
Metal ion blocking Taq DNA polymerase polymerization activity, magnesium ion (Mg 2+) competition deblocking and amplification are adopted, and different mixing proportion comparison tests of Taq DNA polymerase and calcium ion (Ca 2+) are set:
Taq DNA polymerase and calcium chloride are uniformly mixed at a ratio of 10U/mu L:50 mM (denoted as Taq-Ca 1), 10U/mu L:80 mM (denoted as Taq-Ca 2), 10U/mu L:100 mM (denoted as Taq-Ca 3), 10U/mu L:120 mM (denoted as Taq-Ca 4) and 10U/mu L:150 mM (denoted as Taq-Ca 5) at 25 ℃ and left to stand for 30 min to prepare a PCR reaction system, wherein the reaction system comprises the following formula:
system template selection lambda DNA:
Primer I:CGCTCTGGTTATCTGCATCA(SEQ ID No.1);
Primer II:TCACGCAGAGCATCATTTTC(SEQ ID No.2);
the PCR conditions were as follows:
After the reaction is finished, the sample is characterized by agarose electrophoresis, and the result is shown in FIG. 1, FIG. 1 is a graph showing the result of amplification after Ca 2+ with different concentrations is combined with Taq DNA polymerase, and in the PCR amplification, mg 2+ competes for combining with the Taq DNA polymerase, wherein the Taq sample of a control group is Taq DNA polymerase directly added into a system for PCR amplification, the Taq-Ca1, taq-Ca2, taq-Ca3, taq-Ca4 and Taq-Ca5 sample are PCR amplified after the blocking activity Mg 2+ is incubated with CaCl 2 by using Taq DNA polymerase, the graph shows that the experimental group realizes specific amplification, the non-specific amplification is carried out in the control group with unblocked polymerization activity, the ratio of Taq DNA polymerase to calcium ions is 10U/mu L80-100 mM, the non-specific amplification is obviously superior to other experimental groups, the non-specific amplification does not appear in the result graph in the range, and meanwhile, the specific signal of the Taq signal is strong in the range of 10U/mu L, the specific signal is 120U mM mu L, the non-specific signal is not present in the graph, and the optimal signal is not mixed with the Taq signal of 35 mu.35L, although the specific signal is strong, and the optimal signal is not strong in the range of 35 mu.L, although the specific signal is not strong, and the amplification is not good, and the amplification is shown in the optimal signal is 35. Mu.L, although the specific signal is shown in the 5.
Example 2
Metal ion blocking Taq DNA polymerase polymerization activity, competitive deblocking with magnesium ion (Mg 2+), amplification, and comparison test of different magnesium ion (Mg 2+) addition amounts were set:
taq DNA polymerase and calcium chloride are uniformly mixed at 25 ℃ in a ratio of 10U/mu L to 100: 100 mM (marked as Taq-Ca 1) and kept stand for 30: 30min to prepare a PCR reaction system, and the formula of the reaction system is as follows:
system template selection lambda DNA:
Primer I:CGCTCTGGTTATCTGCATCA(SEQ ID No.1);
Primer II:TCACGCAGAGCATCATTTTC(SEQ ID No.2);
the PCR conditions were as follows:
After the reaction is finished, the sample is characterized by agarose electrophoresis, and as a result, referring to FIG. 2, FIG. 2 is a graph showing the result of amplification after the Ca 2+ and the Taq DNA polymerase are combined, different concentrations of Mg 2+ compete for combining with the Taq DNA polymerase during PCR amplification, wherein a control group Taq sample is directly added into a system for PCR amplification, an experimental group Taq-Ca3+ different concentration MgCl 2 sample is subjected to PCR amplification after the blocking activity is incubated with CaCl 2 by using the Taq DNA polymerase and the MgCl 2+ is blocked, 5 mM (4, 5), 10 mM (6, 7), 20mM (8, 9), 30mM (10, 11) and 40mM (12, 13) MgCl 2 are respectively added to 4 to 13 samples. As is evident from the figure, the control group without blocking the polymerization activity is subjected to nonspecific amplification, the amplification effect is close when the addition amount of MgCl 2 is 20mM,30mM and 40mM, the specificity is obviously better than that of 5 mM and 10 mM, and the addition amount of MgCl 2 with the comprehensive specific product is more excellent when the addition amount of MgCl 2 is 20mM, so that the optimal addition amount of Mg 2+ is 20mM. That is, the molar ratio of calcium ion to magnesium ion is not more than 1:1, and the target band signal is strong when the molar ratio of calcium ion to magnesium ion is 1:1 to 1:2, and no nonspecific signal occurs, whereas no nonspecific signal occurs but almost no target band signal occurs when the molar ratio is 1:1 or less (5 mM,10 mM).
Example 3
Metal ion blocking Taq DNA polymerase polymerization activity, metal chelating agent is used for chelating metal ion for blocking and amplification:
taq DNA polymerase and calcium chloride are uniformly mixed at a ratio of 10U/mu L to 100 mM (denoted as Taq-Ca 3) at 25 ℃, and a PCR reaction system is prepared after standing for 30min, wherein the formula of the reaction system is as follows:
selecting lambda DNA as a system template;
Primer I:CGCTCTGGTTATCTGCATCA(SEQ ID No.1);
Primer II:TCACGCAGAGCATCATTTTC(SEQ ID No.2);
the PCR conditions were as follows:
After the reaction is finished, the sample is characterized by agarose electrophoresis, and the result is shown in fig. 3, fig. 3 is a schematic diagram of amplification after Ca 2+ and Taq DNA polymerase are combined and EGTA chelated Ca 2+ is added for deblocking during PCR amplification, wherein a control group Taq sample is formed by directly adding Taq DNA polymerase into a system for PCR amplification, an experimental group Taq-Ca3+ with different concentrations of EGTA sample is formed by using Taq DNA polymerase and CaCl 2 for incubation and blocking activity and then using EGTA with different concentrations of EGTA for PCR amplification, and No. 4 to No. 9 sample are respectively formed by adding 20 mM,30 mM,40 mM,50 mM,60 mM,80 mM EGTA, so that the specific amplification of the experimental group part is obviously realized, and the non-specific amplification of the control group with unblocked polymerization activity occurs. It can be seen from the figure that the molar ratio of calcium ions to chelating agent EGTA is not more than 1:2.5, and if the ratio is exceeded, the nonspecific signal is significantly enhanced, the molar ratio of calcium ions to chelating agent EGTA is 1:1-1:1.5, in which the nonspecific amplification is significantly reduced, and the ratio of Ca 2+ to EGTA is optimal when the ratio is 1:1.
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
Claims (5)
1. A PCR amplification method is characterized in that when a PCR amplification reaction system is prepared, salt containing metal ions is premixed with a DNA polymerase solution for PCR amplification, so that the metal ions are combined with the DNA polymerase, the combination sites of magnesium ions and the DNA polymerase are occupied, the polymerization activity of the DNA polymerase is blocked, magnesium ions are added during PCR amplification to compete for combining with the DNA polymerase combined with the metal ions, the activity of the DNA polymerase is recovered, the combination sequence of the metal ions and the DNA polymerase is lower than the combination sequence of magnesium ions and the DNA polymerase, the DNA polymerase is Taq DNA polymerase, the metal ions are calcium ions, the ratio of the Taq DNA polymerase to the calcium ions is 10U/. Mu.L, the molar ratio of the calcium ions to the magnesium ions is 80-100 mM, and the molar ratio of the calcium ions to the magnesium ions is not more than 1:1.
2. The method of claim 1, wherein the molar ratio of calcium ions to magnesium ions is 1:1-1:2.
3. The method according to any one of claims 1-2, wherein a metal chelating agent is also added during PCR amplification, said chelating agent chelating said calcium ions in a better order than said chelating agent chelating said magnesium ions.
4. A method according to claim 3, wherein the metal ion is a calcium ion and the chelating agent is EGTA, the molar ratio of the calcium ion to the chelating agent being not less than 1:2.5.
5. The method of claim 4, wherein the molar ratio of the calcium ions to the chelating agent is 1:1 to 1:1.5.
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